Plant volatile elicitor from insects

ABSTRACT

The invention relates to fatty acids conjugated to amino acids and their derivatives that elicit the production and/or release of plant volatiles compounds which attract and/or retain beneficial insects and deter herbivorous insect feeding. These conjugates also induce plants to increase production of pharmacologically important compounds such as taxol, increase fragrance of flowers, and increase production of plant essential oil. The invention also relates to methods for isolating the compounds from herbivorous insect oral secretions and to chemically synthesizing the compounds and their active derivatives.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compounds which are isolated from the oralsecretions of beet armyworm caterpillars, to chemically synthesizedcompounds, and derivatives thereof. These compounds induce plants toproduce and/or release a blend of volatile compounds which, for example,can attract and/or retain beneficial insects, increase fragrance inflowers, increase essential oils in plants, such as mint, which aregrown for agricultural production, increase production ofpharmacologically important compounds such as taxol, etc, in plants. Thepresent invention also relates to methods for making the compounds andto their use as effective inducers of useful plant compounds.

2. Description of the Related Art

Arthropod plant pests cause extensive and severe damage to majoragricultural commodities, both in the field and in the greenhouseenvironment. In addition to feeding damage, many of these insects alsotransmit viral diseases. Chemical insecticides are used to controlinsects that damage agricultural commodities such as corn, cotton,barley, beans, citrus, etc. However, recent concerns about insecticideresidues on commodities, resistance of insects to chemical insecticides,hazardous exposure to people who apply pesticides, environmentalcontamination, destruction of natural biocontrol agents such asbeneficial insects, and lack of newly developed insecticides haveincreased the need for alternative control methods. Furthermore, aspests become more resistant to pesticides, more frequent treatments arerequired which compounds the problems identified above. An alternativeto chemical pesticides is the use of biocontrol agents such asbeneficial insects which parasitize and/or eat harmful insects. A usefulway to attract and/or keep beneficial insects on crops is needed inorder to control harmful herbivorous insects and to at least reduce theamount of pesticides needed for the control of herbivores.

Stress-induced reactions in plants are common and the production andtransport of chemicals in response to such stress are proposed tofunction as a direct defense against herbivores and pathogens. Forexample, terpenoids and indole are likely to be defensive compounds orby-products of stress-produced compounds. However, in addition toinitiating direct chemical defenses, herbivore-injured plants benefitdirectly by signaling information into their environment. Thisphenomenon appears to be common; several of the terpenoids released bydamaged corn seedlings are also released by leaves of other plants underattack by caterpillars or mites. The chemical signals, in addition tobeing attractants to natural enemies of the herbivores, may alsofunction as repellents to herbivores. Plants contain a number of organicchemicals which attract pollinating insects or symbiotic organisms andprovide self-defense against pathogens and herbivores (Blechert et al,Proc. Natl. Sci, USA, Volume 92, 4099-4105, May, 1995). Wound damage tothe leaves of plants from a number of families results in the synthesisof proteinase inhibitor proteins at the wound sites as well as in distalleaves. Jasmonic acid and methyl jasmonate are examples of twoplant-derived chemicals which can regulate the expression ofwound-inducible proteinase inhibitor genes. Farmer and Ryan (Plant Cell,Volume 4, 129-134, 1992; Trends Cell Biol., Volume 2, 236-241, 1992;both herein incorporated by reference) show that the signal involved inthe induction of the high molecular weight proteins involves alipid-based transduction system which yields jasmonic acid and itsmethyl ester. It is believed that plant wounding by insects or microbialpathogen attack leads to an interaction of elicitors with receptors thusinitiating the octadecanoic-based pathway from C₁₈ fatty acid linolenicacid to jasmonic acid. Synthetic jasmonic acid also acts as a powerfulinducer of de novo defense protein synthesis, simulating a woundresponse.

Jasmonic acid is a naturally occurring compound identified in plantsfrom at least nine families (Farmer et al, Trends in Cell Biol., supra).Its methyl ester has been used as a perfume fragrance. Jasmonic acid andmethyl jasmonate exhibit diverse biological activities in plants inregulating physiological processes and gene expression. Exogenouslyapplied jasmonates have been found to accelerate senescence, induce thesynthesis of vegetative storage proteins (VSPs) and proteinase inhibitorproteins in leaves, inhibit pollen germination, stimulate ethyleneproduction in tomato fruit, and promote chlorophyll degradation inbarley and anthocyanin production in soybeans. Jasmonates arecyclopentanone compounds (See FIG. 2a) which are commonly presentthroughout the plant kingdom. The structures and bioactivities ofjasmonates are thoroughly reviewed by Hamberg and Gardner (Biochimica etBiophysica, Volume 1165, 1-18, 1992) and Parthier (Biochimica etBiophysica Acta, Volume 104, 446-454, 1991), the contents of each ofwhich are herein incorporated by reference herein. Jasmonates have beendescribed as exerting a wide range of differing effects on virtually allplants. These effects range from inhibition to promotion of plantprocesses. As Parthier describes, the effect exhibited on the plant mayeven be concentration dependent, with some processes stimulated at lowerconcentrations but inhibited at higher concentrations. Jasmonates havebeen reported to regulate growth patterns in soybean, induce geneexpression during zygote embryogenesis in Brassica, cause tendrilcoiling in Bryonia and cell-cycle-dependent disruption of microtubles intobacco cells. Jasmonates have also been shown to inhibit seedgermination and seedling growth, stimulate seed germination (at lowerconcentrations), promote seed dormancy breaking, and promote leafsenescence. Farmer et al (1992, supra) also show that octadecanoidprecursors of jasmonic acid; linolenic acid, 13(s)-hydroperoxylinolenicacid and phytodienoic acid can also act as signals for proteinaseinhibitor induction in tomato leaves when applied to leaf surfaces.Tazaki (Japanese kokai 2-92220 (A), published Apr. 3, 1990, patentapplication no. 63-242432, filed Sep. 29, 1988), Yoshihara et al, Agric.Biol. Chem., Volume 53, 2835-2837, 1989), Matsuki et al (Biosci,Biotech, Biochem., Volume 56, 1329-1330, 1992) and Koda et al (PlantCell Physiol., Volume 29, 969-974, 1988) all disclose treating potatostem fragments with jasmonates in culture to induce tuber formation.These compounds are considered to be endogenous regulators of plantresponses.

Corn plants attacked by caterpillars release volatile terpenoids. Theparasitic wasp Cotesia marginiventris, a generalist larval parasitoidthat attacks many different lepidopterous species, locates caterpillarhosts in response to terpenoid release. In locating the caterpillar, thewasp injects an egg into it. In response the caterpillar immediatelyslows its feeding habit. Later, the wasp egg develops into a larvae thatfeeds internally on the caterpillar. Tumlinson et al (C&EN, Sep. 7,1992) show that the oral secretions from caterpillars, which contain acombination of digestive fluids and saliva, induce local terpenoidrelease when applied to scratched surfaces of leaves on corn plantseedlings. The plant response cannot be induced by artificial damageunless the damaged sites are treated with the regurgitant of larvae(Florida Entomologist, Volume 74(1), 42-50, March, 1991). The terpenoidsare released from the scratched leaves as well as from undamaged leaves.This indicates that the release of terpenoids is systemic and not justlocalized to the damaged leaves. Undamaged plants are far lessattractive to beneficial insects than plants damaged overnight by beetarmyworm (BAW, Spodoptera exigua Hubner) larvae. The strongestattraction is observed when larvae are put back on an already damagedcorn seedling. Furthermore, the corn seedlings on release of theterpenoids and indole become less palatable to the BAW larvae.

While it is known that oral secretions from herbivores and plant-derivedprecursors of jasmonic acid induce the release of terpenoids by plants,other factors, especially herbivore-derived, have not been identified,isolated, and/or synthesized. Currently, the compounds of the presentinvention are only available by using crude preparations of oralsecretions of herbivorous insects. It is desirable to be able tochemically synthesize these compounds and active derivatives in order tomake their use economically feasible. The only substance from insectoral secretions found to induce volatile chemical release by plants thusfar is beta-glycosidase from Peris brassicae larvae(Mattiacci et al,PNAS, USA, Volume 92, 2036, 1995). The present invention providesnon-enzymatic herbivore-derived compounds, synthetic compounds andderivatives, different from related art compounds. The compounds of theinvention induce plants to produce and/or emit volatile compounds, suchas for example, compounds which attract natural enemies of herbivores,increase production of pharmacologically important compounds, increasefragrance of flowers, increase production of plant essential oils, etc.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide novelelicitors and their derivatives of plant volatile terpenoids and indole.

Another object of the present invention is to provide methods forobtaining the elicitors and their derivatives either by synthesis orextraction from oral secretions of herbivores.

A still further object of the present invention is to provide a methodfor treating plants by administering effective amounts of an elicitor orits derivative in order to stimulate volatile compound production and/orrelease in plants.

Another object of the present invention is to provide a compositioncomprising a novel elicitor or its derivative in amounts effective toincrease the production and/or release of volatile compounds in plants .

Further objects and advantages of the invention will become apparentfrom the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of N-(17-hydroxylinolenoyl)glutamine.

FIG. 2a depicts the octadecanoid pathway found in plants.

FIG. 2b depicts the synthetic pathway to prostaglandins andleukotrienes.

FIG. 3 is a graph showing chromatographic profiles of volatiles releasedby corn seedlings that were placed for 12 hours in vials with distilledwater or vials with a 10-fold dilution of beet armyworm (BAW)regurgitate. The identities of the various compounds have beendetermined previously (Turlings et al., J. Chem. Ecology, Volume 17,2235-2251, 1991). 1, (Z)-3-hecen-1-yl acetate; 2, linalool; 3,(3E)-4,8-dimethyl-1,3,7-nonatriene; 4, indole; 5,alpha-trans-bergamotene; 6, (E)-beta-farnesene; 7, (E)-nerolidol; and 8,(3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. Internal standard(IS) is nonyl acetate (600 ng).

FIG. 4 is a graph showing average amounts (ng/2 hr.) of four replicatesof volatiles collected from three corn seedlings that have been treatedwith either 15 microliters of BAW regurgitate/seedling on the damagesite (Regrugitate), 15 microliters regurgitate equivalents of purenatural elicitor (Natural elicitor), or an equal amount of just buffer(Damaged), and undamaged control plants (Undamaged).

FIG. 5 shows the synthetic scheme for 17-hydroxylinolenic acid.

FIG. 6 is a graph showing average of four replicates of relative releaseof volatiles collected under 2 hours from three corn seedlings that hadbeen treated with 15 microliters of regurgitate equivalents/seedling(300 picomole/plant) or 45 microliters of regurgitateequivalents/seedling (900 picomole/plant) of, the L-glutamine derivativeof synthetic elicitor (L-syn), the D-glutamine derivative of syntheticelicitor (D-syn), the sodium form of synthetic 17-hydroxy-linolenic acid(17 OH), L-glutamine (L-GLN), D-glutamine (D-GLN) and just buffer (CON).The combined amount in nanogram of Caryophyllene,alpha-trans-bergamontene, (E)-beta-farnesene, (E)nerolidol, and(3E,7E)-4, 8, 12-trimethyl-1, 3, 711, tridecatetraene was used tocalculate the release relative to that of corn seedlings treated with ISmicroliters regurgitate (300 picomole/seedling of natural elicitor).

FIG. 7 is a graph showing average of three replicates of relativerelease of volatiles collected under 2 hours form three corn seedlingsthat had been treated with 15 microliters of regurgitateequivalents/seedling (300 picomoles/plant) or 45 microliters ofregurgitate equivalent/seedling (900 picomoles/seedling) of, L-glutaminederivative of linolenic acid (L-Gln-Lin), the D-glutamine derivative oflinolenic acid (D-Gln-Lin), natural elicitor (Natural), and just buffer(Control). The combined amount in nanogram of Caryophyllene,alpha-trans-gergamotene, (E)-beta-farnesene, (E)-nerolidol, and(3E,7E)-4, 8, 12-trimethyl-1, 3, 7, 11, tridecatetraene was used tocalculate the release relative to that of corn seedlings treated with 15microliter regurgitate (300 picomole/seedling of natural elicitor).

FIG. 8 is a graph showing the relative release of volatiles (fourreplicates) collected over a period of about 2 hours from three cornseedlings that had been treated with 5, 17, 50, 170 and 500picomoles/plant of natural elicitor (Natural) and the L-form ofsynthetic elicitor (Synth), and treated with just buffer (Buffer). Thecombined amount in nanograms of Caryophyllene, alphatrans-bergamotene,(E)-beta-farnesene, (E)-nerolidol, and (3E,7E)-4, 8, 12-trimethyl-1, 3,7, 11, tridecatetraene was used to calculate the release relative tothat of corn seedlings treated with 15 microliters regurgitate (300picomoles/seedling of natural elicitor). The same capital lettersindicate no significant difference between doses of natural elicitor andthe same lower case letters indicate no significant difference betweendoses of synthetic elicitor (paired t-test, p<0.05). At no dose wasthere a significant difference between natural and synthetic elicitor.

DETAILED DESCRIPTION OF THE INVENTION

It is found that the defensive reaction of plants, whereby plantvolatiles are induced by insect herbivores, is triggered by contact of asubstance in the oral secretion of insect herbivores with damaged planttissues. The isolation, identification and synthesis of a compound fromthe oral secretion of beet armyworm (Spodoptera exigua Hubner)caterpillars that induces corn (Zea mays L.) seedlings to release thesame blend of volatile compounds as released when they are damaged bycaterpillar feeding is the first identification of a non-enzymatic plantvolatile elicitor from insects. The production of the compounds of thepresent invention is not diet related and, thus, does not originate fromplants (Turlings et al, J. Chem. Ecol., Volume 19, 411, 1993; hereinincorporated by reference in its entirety). The oral secretion ofinsects fed on an artificial diet is as active as that from those fed onplants. Both corn and cotton respond to BAW damage and to the oralsecretions of BAW applied to damaged leaves by producing and releasingterpenoid compounds and indole. While some compounds, like indole,ocimene and farnesene, for example, are released by both plants, othercompounds are unique to each plant. This is most likely the result ofdifferences in plant chemistry. Both plants respond systemically to thecompounds of the present invention by releasing induced volatiles fromundamaged leaves of injured plants (Turlings et al, PNAS, USA, Volume89, 8399, 1992 and Rose et al, Plant Physio., Volume 111, 487, 1996;both herein incorporated by reference in their entirety). Thesecompounds are related to eicosapentaenoic and arachidonic acids from thefungus Phtophthora infestans that elicit the production of fungitoxicsesquiterpenes in potato (Bostock et al, Science, Volume 212, 67, 1981).These compounds, isolated or synthetic, and active derivatives arerelated to eicosanoids and prostaglandins that play important regulatoryand signaling roles in insects and other animals and the components ofthe octadecanoid signaling pathway in plants (See FIGS. 2a and 2b). Inplant systems studied so far, biosynthesis and release of volatilecompounds appear to be induced by jasmonic acid, which is a product ofthe octadecanoid signaling pathway. Jasmonates have also been reportedto stimulate numerous other physiological and defensive processes inplants. Furthermore, the amino acid conjugates of jasmonic acid areinvolved in physiological and developmental processes in many plants.Therefore, the presence of an elicitor such as an octadecanoateconjugated to an amino acid suggests that the elicitor molecule in someway interacts with or amplifies the octadecanoid pathway in herbivoredamaged plants.

Oral secretions from at least five species of lepidopterous larvae andof grasshoppers induce corn seedlings to release the same blend ofvolatiles (Turlings, 1993, supra). The grasshopper oral secretionappears to be more active than that of caterpillars in inducing cornseedlings to release volatiles.

It has now been discovered that C₁₆ -C₂₄ fatty acids conjugated with anamino acid, whether isolated from the regurgitate of herbivorous insectsor chemically synthesized, induce plants to produce and/or releasevolatile compounds. These released compounds can have many activities,such as for example, attracting and/or retaining beneficial insects oncrops and deterring herbivorous insect feeding. The released compoundsare, for example, the same compounds released when the plants aredamaged by herbivorous insect feeding.

The search for new compounds possessing useful biological activitiesrequires that the new compounds and known compounds, especially naturalproducts and their derivatives, be synthetically prepared. Syntheticpreparation of natural compounds allows economical use of theseproducts. The process of the present invention is particularly usefulfor the manufacture of C₁₆ -C₂₄ fatty acids conjugated with amino acids.The invention describes the preparation of these conjugated compoundswhich have biological activity in stimulating plants, such as, forexample, corn and cotton, to synthesize terpenes and other compounds.The method for synthesizing the specific acid conjugates is importantbecause it allows large quantities of the compounds to be produced in arelatively short period of time as compared to the labor intensivemethod of extracting the acids from the oral secretions of herbivorousinsects.

To isolate and identify the active compound from beet armywormcaterpillar, oral secretions of the caterpillar are centrifuged toremove solid material and the supernatant is filter sterilized. Theproteins are precipitated from the supernatant and the precipitateremoved by centrifugation. The resulting supernatant is fractionated byreverse phase solid phase extraction using water, acetonitrile in water(about 50/50) and acetonitrile. The resulting aliquots are furtherseparated by liquid chromatography. The collected fractions arebioassayed to identify the fraction(s) containing the active material.The active material is purified by a series of chromatographic stepsincluding reverse phase HPLC and liquid chromatography to obtain theactive compound at about 99% purity by HPLC. The purified compound isidentified as a 17-hydroxylinolenic acid glutamine conjugate (FIG. 1)The C₁₆ to C₂₄ fatty acids conjugated with an amino acid refers to acombination of a C₁₆ to C₂₄ fatty acid and a D- or L-amino acid. Theterm fatty acid is meant to include C₁₆ to C₂₄ carboxylic acids.Carboxylic acids, for the purposes of this application, includealiphatic acids and their unsaturated and saturated, alkylated,substituted, oxidized or hydroxylated derivatives. Examples of fattyacids of the present invention include, for example, hexadecanoic acid,octadecanoic acid, eicosanoic acid, tetracosanoic acid, palmitoleicacid, oleic acid, linoleic acid, linolenic acid, arachadonic acid, etc.For the purposes of the present invention, C₁₆ to C₂₄ acids can bealkylated, substituted, oxidized, or hydroxylated derivatives.Substituents on the aliphatic chain may include aryl groups, aminogroups, formyl groups, or other heteroatom groups. The compounds of thepresent invention include, but are not limited to,17-hydroxylinolenoyl-L-glutamine, 17-hydroxylinolenoyl-D-glutamine,linolenoyl-L-glutamine, linolenoyl-D-glutamine, andN-(17-hydroxy-linolenoyl)glutamine which is isolated from beet armywormoral secretions. The most preferred is the unsaturated C₁₈ carboxylicacid, 17-hydroxylinolenic acid which is conjugated with L-glutamine.

The term amino acid for the purposes of this invention is meant toinclude any alpha-amino acid including D- or L-forms. Examples of aminoacids useful in the present invention include glutamine, asparagine,alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, tryptophan, glycine, serine, threonine, cysteine,tyrosine, aspartic acid, glutamic acid, lysine, arginine, histidine,etc. L-glutamine is most preferred. The amino acid is conjugated withthe fatty acid via an amide bond with the alpha amino group.

The synthesis of the compounds (FIG. 5) of the invention begins with theproduction of 1-ethoxyethyloxy-8-benzoyl-3,6-nonadiyne (1) by reactingethoxyethyl ether of 3,6-heptadiynol with an aldehyde such asacetaldhyde, for example, and benzoyl chloride. Any aldehyde can be usedthat will result in a fatty acid as defined by the present application.Known compounds, such as for example, ethoxyethyl ether of3,6-heptadiynol, acetaldehyde, and benzoyl chloride can be purchased orprepared by any known method in the art. The ethoxyethyl ether group isremoved from compound I to form 3,6-nonadiyn-1,8-diol, 8 benzoate whichis reacted with quinoline and Lindlar catalyst to form8-benzoyl-Z,Z-3,6-nonadiene-1-ol (II). Compound II is sulfonated andthen iodinated. The mixture is then treated with triphenylphosphine toform 1triphenylphosphonium-8-benzoyloxy-Z,Z-3,6-nonadiene iodide (III).Compound III is reacted with hexamethyldisilazide to form thecorresponding ylide. The ylide is reacted with a CHO(CH₂)_(n) COOCH₃,wherein n=about 5-13, to form methyl 17-benzoyloxymethyllinolenate (IV).The benzoyl group is then removed to obtain a hydroxy fatty acid. Thefatty acid can be hydrogenated to obtain a saturated compound usingmethods standard in the art. The amino group is then conjugated to thefatty acid via an amide bond with the alpha amino group.

More specifically, to chemically prepare the compounds of the invention,the C₁₆ -C₂₄ fatty acids are prepared first, and isolated. The isolatedfatty acid is then conjugated to the amino acid. The first step of theprocess produces a 1-ethoxyethyloxy-8-benzoyl-3,6nonadiyne [I] byreacting ethoxyethyl ether of 3,6 heptadiynol in an anhydrous etherealsolvent, such as, for example, tetrahydrofuran with an organometallicbase, such as, for example, ethylmagnesiumbromide in an anhydrousethereal solvent, tetrahydrofuran, for example, at about 0° C.Approximately 1 ml of acetaldehyde, or any other aldehyde, is added tothe mixture. The mixture is then treated with an anhydrous tertiaryamine base, such as for example, pyridine, followed by benzoyl chlorideto acylate the resulting alcohol. This mixture is stirred forapproximately 1 hour and is then treated with a saturated ammoniumchloride and extracted into a non-polar solvent, such as, for example,diethyl ether. The organic phase is separated, washed in brine and driedover anhydrous inorganic salt, such as, for example, MgSO₄. For thepurposes of this application, brine is defined as a saturated sodiumchloride solution. The dried preparation containing I is reconstitutedin an alcohol solvent, such as, for example, methanol and treated withan organic acid, such as for example, p-toluenesulfonic acid to removethe ethoxyethyl ether group. This is allowed to react until almost nostarting materials remain. The mixture is treated with a mild inorganicbase, such as for example, NaHCO₃, and the solvent is removed in vacuo.The residue is taken up in a non-polar solvent, such as for example,diethyl ether and purified by liquid chromatography over silica gel toobtain 3,6-nonadiyne-1,8-diol, 8-benzoate. The solvent is removed invacuo and the residue taken up in an alcoholic solvent, such as, forexample, ethanol, containing quinoline and Lindlar catalyst and stirredat about room temperature until hydrogen uptake ceases. The product isisolated by liquid chromatography through celite and the solvent removedin vacuo. The residue is taken up in a non-polar solvent, such as, forexample, diethyl ether, washed with dilute aqueous acid, such as, forexample, 10% HCL, and dried over an anhydrous inorganic salt, such as,for example, MgSO₄. The dried residue is taken up in methylene chlorideand this contains compound II.

1-Triphenylphosphonium-8-benzoyloxy-Z,Z-3,6-nonadiene, iodide [III] isproduced by reacting the methylene chloride preparation containing IIwith a tertiary amine, such as, for example, triethyl amine and asulfonyl chloride, such as, for example, methanesulfonylchloride toproduce the corresponding sulfonate. This is then taken up in anon-polar solvent, such as, for example, diethyl ether, washed, driedand solvent removed in vacuo. The residue is taken up in a low molecularweight ketonic solvent such as acetone and stirred with an excess ofsodium iodide for approximately 2-6 hours. The solvent is removed invacuo, and the residue taken up in a non-polar solvent such as diethylether and washed with a dilute solution of sodium thiosulfate and driedover an anhydrous inorganic salt such as MgSO₄ For the purposes of thisapplication, a dilute solution of sodium thiosulfate includes solutionsof about 5-20% sodium thiosulfate. The solvent is removed in vacuo andthe residue is taken up in acetonitrile and treated withtriphenylphosphine and refluxed for at least about 8-12 hours. Thesolvent is removed and the brittle foam is taken up in a polar etherealsolvent, such as for example, tetrahydrofuran and cooled toapproximately -80° C. under an argon atmosphere. This contains compoundIII.

The mixture containing III is then treated with sodiumhexamethyldisilazide in order to form the corresponding ylide and then asolution of CHO(CH₂)_(n) COOCH₃ in a polar ethereal solvent, such as forexample, tetrahydrofuran is added and the mixture is stirred for atleast about 8-12 hours and warmed to approximately room temperature. Forthe purposes of this invention n=about 5-13. Compound IV is extractedfrom this mixture by the addition of a saturated ammonium chloridesolution and a non-polar solvent such as diethyl ether. The organicphase is removed and contains Methyl, 17-benzoyloxy(CHO(CH₂)_(n) COOCH₃)which is compound IV.

The benzoyl group and the methoxy group are removed by treating themixture containing IV with aqueous LiOH, and tetrahydrofuran at aboutroom temperature for at least about 8 hours in order to obtain a hydroxyfatty acid [V]. The compound is extracted using standard methods in theart. A saturated product can be obtained by hydrogenating compound Vusing methods standard in the art.

To obtain the amino acid conjugate, compound V is reacted with thelithium salt of the desired L- or D-amino acid. The lithium salt of theamino acid is dispersed in an aprotic polar solvent such as for exampleDMF to which is added water and freshly distilled triethylamine. In asecond container compound V is dissolved in an aprotic polar solvent,such as for example DMF, followed by hydroxybenzotriazole. 1-ethyl-3(Dimethylaminopropyl) carbodiimide is added and the solution stirred forabout 1 minute and then the contents of the container containing theamino acid is added and stirred overnight at about room temperature tocouple the two compounds. The conjugate is extracted and purified bycolumn chromatography using methods well established in the prior art.

The compounds of the present invention can be sprayed on crop plants inthe field under conditions which induce plants to produce and/or releasevolatile chemicals in order to attract beneficial insects which arenatural enemies of the herbivorous pests. The compounds are sprayed oncrop plants when an outbreak of insect pests is first detected torecruit beneficial insects earlier and in larger numbers than occursunder natural conditions. In addition, "factory" reared parasiticinsects or other beneficial insects can be released at the time ofspraying in order to retain the released insects in the target areauntil they find the pest insects. The compounds of the present inventionare mixed with a surfactant, such as, for example, Silwet L-77(polyalkylenoxide modified polydimethylsiloxane; Union Carbide,Tarrytown, N.J.), which ensures that the compound of the presentinvention is carried into the plant. Silwet L-77 is used to carry manysubstances into plant leaves including plant hormones like gibberellinand cytokinin (Zidack et al, Biological Control, Volume 2, 111-117,1992; herein incorporated by reference). It reduces the surface tensionto about 20 dynes cm⁻¹ allowing the liquid to enter leaf stomata. Timingof spray application is coordinated with day length such that plantswould be sprayed in late afternoon to allow overnight incubation so thatplants would produce and/or release volatiles during the nextphotophase. In some instances, depending on the plant species, anincubation period of greater than 24 hours might be required to induceplant volatile production and/or release. For example, in laboratoryconditions, soybeans respond more slowly to the application of thecompounds of the present invention than do corn seedlings.

Another method of using the compounds of the present invention includesspraying plants with the compounds of the present invention insurfactant solutions, as described above, before the plants are attackedby pests in order to immunize the plants to insect attack. It is wellestablished that systemic acquired resistance occurs in plants whereinthey become resistant to subsequent attack after undergoing an initialattack by insects or pathogens. Plants attacked by beet armyworm larvaeor treated with beet armyworm larval oral secretions will releasevolatile compounds from undamaged leaves (Turlings et al, 1992, supra,Rose et al, 1996 supra; both herein incorporated by reference in theirentirety). By spraying plants with these compounds the biochemicalmechanisms are primed to respond more quickly and strongly if they aresubsequently attacked by pests. The plants will not sustain the initialdamage as they would if initially attacked by insects.

The elicitor compounds of the present invention can be used to increasethe production of essential oils by mint and other crops commerciallygrown for the production of essential oils. Compounds are mixed with asurfactant, as described above, such as Silwet-77 that will allow theelicitor to penetrate the leaf surface through the stomata.

The elicitor compounds of the present invention can be suppliedhydroponically through the roots of plants by adding the elicitors tothe hydroponic solutions used to feed plants. Since these compounds haveboth a polar amino acid group and a non-polar lipid moiety, they aresoluble in a wide variety both non-polar and polar solvents, such as,for example, water and organic solvents. By allowing the plants to takethe compounds up through the roots they can be induced to producecertain compounds of industrial and medicinal value or to produceincreased amounts of certain compounds that will enhance their flavor.Furthermore, the elicitor compounds of the present invention can beadded to plant cell cultures or tissue cultures to induce the productionof increased amounts of compounds of industrial or medicinal value. Forexample, an elicitor of the present invention can be added to cellcultures of taxol-producing plants, such as Taxus media, to increase theproduction of taxol, an important cancer fighting agent.

The elicitor compounds of the present invention can be used to increasethe perfume smell of cut flowers and floral arrangements. The compoundscan be dissolved in a solution such as approximately 50 mM sodiumphosphate/citrate buffer or a similar buffer, and the freshly cut stemsof the flowers immersed in a container or vase containing the solutioncontaining the elicitor compounds of the present invention and theflowers take up the solution through the cut stems. Water is added asneeded to maintain the flowers in fresh condition. After standingovernight in the solution, flowers will begin to release increasedamounts of perfume when they are exposed to light, especially sunlight.

The following examples are intended to further illustrate the inventionand are not intended to limit the scope as defined by the claims.

EXAMPLE 1

To isolate N[17-hydroxylinolenoyl]-glutamine (Example 1), oralsecretions were collected by gently squeezing 3rd-5th instar BAWcaterpillars that had been fed on corn seedlings, causing them toregurgitate. The liquid oral secretion is drawn into a glass capillarytube connected to a small glass bottle under vacuum. The oral secretionis centrifuged at approximately 16,000 g for about 30 minutes toeliminate solid material followed by a sterile filtering of thesupernatant through an approximately 0.22 μm membrane filter (Millex GV,Millipore, Bedford, Mass.). An equal amount of approximately 50 mM,about pH 3.3 phosphate buffer is added and precipitated proteins removedby centrifugation. An approximately 0.5 ml aliquot of regurgitate inbuffer is fractionated on a reverse phase solid-phase extractioncartridge. The aliquots are put on an activated SPE octadecyl, 6 mlcartridge (Bakerbond, J. T. Baker, Phillipsburg N.J.). A first fractionis collected by allowing about 2 ml of water to flow through the columnby gravity flow. Two more fractions are collected by successive elutionwith about 2 ml of approximately 50% acetonitrile in water (fraction 2)and about 2 ml of about 100% acetonitrile. All fractions are vacuumconcentrated to near dryness (Speed Vac rotary concentrator, SavantInstruments, Farmingdale, N.Y.) and redissolved in about 0.5 ml ofapproximately 50 mM, about pH 8 buffer, prior to being bioassayed in themanner of Turlings et al (J. Chem. Ecol., Volume 19, page 411, 1993;herein incorporated by reference). The total activity of thechromatographed oral secretion elutes from the reverse phase cartridgewith the approximately 50% acetonitrile in water indicating a moleculeof medium polarity. To bioassay the oral secretion, about 45 μl of BAWregurgitate is concentrated to dryness and dissolved in about 1500 μl ofan approximately 50 mM sodium phosphate/citrate buffer about pH 8. Thesolution is divided into three about 500 μl portions in 1 ml glassvials. At about 9 to 11 pm, a corn plant that had spent at least about 1hour in darkness is cut above the root with a razor blade andtransferred to each vial and allowed to suck up the approximately 500 μlaliquots of crude regurgitate or chromatography fractions for about 12hours in complete darkness. Three seedlings for each treatment are puttogether in a volatile collection chamber (Turlings et al, 1991, supra).Purified and humidified air is drawn for about 2 hours through thechamber at about 300 ml/min and through a polymer adsorbent (super q80/100 Cat. No. 2735, Alltech Associates, Deerfield, Ill.). Theadsorbent is removed from the chamber and extracted with about 150 μlmethylene chloride and approximately 600 ng of the internal standardnonyl acetate in about 30 μl methylene chloride was added. The extractis analyzed by capillary gas chromatography (Turlings et al, J. Chem.Ecol., Volume 17, 2235, 1991; herein incorporated by reference). Theresults are shown in FIG. 3.

The active material from solid phase extraction is further fractionatedon reverse phase HPLC using a LDC 4100 pump with SM5000 diode array UVdetector (LDC Analytical, Riviera Beach, Fla.), monitoring UV detectionfrom about 190-360 nm. The reverse phase column (Waters Nova Pac C 18 4μ ID×150 mm column, Waters, Milliford, Mass.) is eluted with a solventgradient from about 0 to 25% acetonitrile in water in about 15 minutesfollowed by an increase to about 100% acetonitrile in about 15 minutes,using a flow of approximately 1 ml/min. The eluate is collected in about2 ml fractions. Approximately all the activity is contained in the about7-9 ml fraction. This fraction is further separated on a second reversephase HPLC column with different selectivity (YMC 18 ODS-AQ S-5 200 Å,46 mm ID×250 mm (YMC Co., ltd. Kyoto, Japan) using the same solventgradient. Almost all the active material elutes in the approximately20-22 ml fraction. This fraction contains two overlapping peaks whichcould be separated on the same column using a solvent gradient ofapproximately 20 to 60% acetonitrile in water over about 20 minutes.This component, with maximum UV adsorption at about 200 nm, eluting fromthe final column is active and its biological activity is equivalent tothat of the original crude regurgitate. When this fraction containingthis component is lyophilized, redissolved in about pH 8phosphate/citrate buffer, and reanalyzed on the final HPLC system(reverse phase HPLC column with different selectivity, supra) theretention volume of the active compound is reduced by about 50%.However, when the pH is adjusted to about 7, the component regained itsoriginal retention volume. This strongly indicates that the activecompound is a weak acid, which is confirmed by extraction of the activematerial into CH₂ Cl₂ from an acidified (approximately pH 3) aqueoussolution, but not from an aqueous solution at about pH 8.

An approximately 2 ml aliquot of the active HPLC fraction isconcentrated to dryness under vacuum. The sample is redissolved inapproximately 2 ml water. Approximately 100 μl acetic acid andapproximately 2 ml methylene chloride are added and the mixture shakenfor about 5 minutes. The water and organic phase are separated andconcentrated to approximately dryness under vacuum. Bioassay of thefractions redissolved in approximately pH 8 buffer show that about allbiologically active material to be present in the organic phase and HPLCanalysis, as described above, shows the peak to be present in thisfraction. Extracting approximately all biological activity from theorganic phase back into pH 8 buffer strongly indicates lipid characterand an acidic functional group.

About a 0.5 ml aliquot of the methylene chloride fraction is put on anactivated 3 ml 10SPE diol cartridge (Bakerbond, J. T. Baker,Phillipsburg, N.J.). The column is rinsed with a first fraction of about3 ml methylene chloride followed by approximately 2 ml of methanol. Theactive component elutes from this column with the methanol.Rechromatography of the active component on the final reverse phase HPLCcolumn indicates that it is greater than about 99% pure by HPLC.

The biological activity of the pure component is confirmed by applyingit in a buffer solution to artificially damaged leaves on corn plants.This treatment resulted in the same induced production of characteristicvolatiles as treatment with BAW regurgitate. The results are shown inFIG. 4. Application of only buffer on an artificially damaged site didnot result in release of volatiles.

EXAMPLE 2

The active compound was identified by mass and infrared spectroscopy andby chemical transformations. Up to about 4 μl of active material (up toabout 40 ng) in an approximately 50% acetonitrile in water solution andabout 1 μl of trifluoroacetic acid is added to a glycerol matrix andanalyzed with Fast atom bombardment mass spectroscopy (FAB-MS), on a VGZagspec (VG Analytical, Fisons Instrument, Manchester, England). Toobtain sodium aducts, the TFA is substituted with approximately 1 μl ofan approximately 1 M sodium chloride solution. High resolution massmeasurements are obtained by adding polyethylene glycol (about 1 μl)with an average mass of about 400 Dalton (PEG 400) to the glycerolmatrix to give reference ions of known mass for an exact calibration ofthe mass scale. Possible elemental compositions are established,allowing a limitation of a maximum of approximately 30 C, a maximum ofapproximately 8 O, and minimum of approximately 2 O. The number ofnitrogens could be either about 2, 4, or 6. The mass window for thecalculations was limited to an error of about 10 milli mass units.Daughter ion spectra are obtained from samples in the same FAB matrix asabove using a high resolution MS\MS tandem four-sector mass spectrometer(Jeol HX/HX110A, Tokyo, Japan). The nitrogen collision gas is adjustedto give an approximately 60% reduction in intensity of mother ion andthe resulting pattern of daughter ion analyzed in a second highresolution mass spectrometer.

The FAB-MS analysis produces diagnostic peaks at m\z 423.280 (M+H)⁺ inthe positive ion mode, and m/z 421.2733 (M-H)⁻ in the negative ion mode.The addition of sodium chloride to the FAB matrix results in reducedintensity of the m/z 423.280 and the appearance of strong m/z 445.2628(M+H)⁺. These results confirm that the active component is a weak acidwith a molecular weight of 422.274 Dalton for the neutral molecule inacid form. The C₂₃ H₃₈ N₂ O₅ (422.278 Dalton) is the only possibleelemental composition.

Daughter ions of the sodium salt, m/z 445, obtained by FAB MS include adominating ion at m/z 427 (445-18), while daughter ions of M/Z 423 gavea strong 405 (423-18), both indicating a loss of water. The lower massregion shows a characteristic pattern of m/z 147, 130, 129, 101, 84, 67,56, which resembles the electron impact (EI) mass spectra of bothglutamine and glutamic acid, but is more consistent with that ofglutamine. (National Institute of Standards and Technology,Gaithersburg, Md., Mass spectral library). Characteristic ions forglutamine are m/Z 130, 129, 101, 84 (base peak) and 56. Subtraction ofglutamine, linked via an ester or amide bond, gave C₁₈ H₃₀ O₃, as anelemental composition of the second part of the molecule, which isconsistent with a hydroxy C18 acid with three double bonds.

For gas chromatographic-mass spectral (GC/MS) analysis, active materialto an equivalent of approximately 100 μl regurgitant is methanolyzedwith methanol/acetic acid anhydride following the procedures of J. M. L.Mee, Biomed. Mass Spectrom., Volume 4, 178, 1977, herein incorporated byreference. The sample is concentrated to dryness and approximately 50 μlof dry methanol and approximately 50 μl acetic anhydride is added. In anitrogen atmosphere, the sample is heated to about 100° C. for about tenminutes and concentrated to dryness by a gentle N₂ blowing.Approximately 50 μl methylene chloride is added and the sample isanalyzed by GC/MS. A Finnigan TSQ 700 mass spectrometer (Finnigan MAT,San Jose, Calif.) is combined with a HP 5890 gas chromatograph (HewletPackard, Palo Alto, Calif.). Helium is used as a carrier gas and methaneis used as the reaction gas for chemical ionization. A polar capillarycolumn (OV351, 25 m×0.25 ID, Scandinaviska Genetec, Kungsbacka Sv) isheld at about 60° C. for approximately three minutes and thentemperature programmed about 10° C./min to about 250° C. and held atthat temperature for approximately 18 minutes. Splitless injectionoccurs at about 225° C.

This reveals two prominent peaks. Chemical ionization (CI)/MS analysisof the first of these peaks with a retention time of approximately 21.05minutes reveals a prominent (M+1)⁺ ion at m/z 144 and electron impact(EI)/MS analysis reveals a molecular ion at m/z 143 and diagnostic ionsat m/z 84 (base peak), 56, and 41 identifying it as the methyl ester ofpyroglutamate, which by congruence in GC retention time and massspectrum with the product of glutamine treated in the same way confirmsthe presence of glutamine. The CI mass spectrum of the second peak (Rtabout 27.55 min), contains a very weak m/z 309 (M+1)⁺ ion and apredominant ion at m/z 291 due to loss of water (M+1-18)⁺. Loss ofmethanol gave an ion at m/z 277 (M+1-32)⁺ and loss of both water andmethanol gives an ion at m/z 259 (M+1-18-32)⁺. The EI spectrum of thesame peak shows no molecular ion but a strong n/z 290 due to loss ofwater (M-18)⁺, and a fragmentation pattern of ions characteristic for astraight chain unsaturated hydrocarbon. These results are consistentwith the methyl ester of a hydroxy acid. A smaller peak in thechromatogram has retention characteristics and a mass spectrumconsistent with the acetate of the same hydroxy acid methyl ester.

Fourier transform infrared analysis was performed using a Fouriertransform Infrared detector 5965B and 5890 Capillary GC (Hewlet Packard,Palo Alto, Calif.). Nitrogen is used as the carrier gas. A non-polar DB1column (25 m×0.35 mm ID, J&W Scientific, Folsom, Calif.) was held atapproximately 60° C. for about three minutes and then temperatureprogrammed to about 10° C./min to about 250° C. and held at thattemperature for approximately 18 minutes. Splitless injection at about225° C. Hydroxy acid methyl ester, Rt. about 25.38 minutes.

Fourier transform infrared analysis of the hydroxy acid methyl esterpeak from GC produces a weak, broad absorption at about 3646 cm⁻¹indicating an alcohol and absorption bands at about 3019, 2935, 2865cm⁻¹, typical for a straight chain non-conjugated unsaturatedhydrocarbon. The intensity of the approximately 3019 cm⁻¹ peak relativeto the others indicates three cis double bonds and the absence of a peakin the area of about 960 to 980 cm⁻¹ indicates no trans double bonds arepresent. An intense absorption at about 1758 cm⁻¹ confirms the presenceof a methyl ester.

These results all confirm that the molecule consists of two subunits,glutamine and a hydroxy C₁₈ acid with three cis bonds. These twocomponents could be attached in three ways to give the right elementalcomposition. However, only an amide bond between glutamine and the acidmoiety of the hydroxy acid would result in a free hydroxy as indicatedby the FAB/MS experiments without also giving a free amine for whichthere is no evidence.

EXAMPLE 3

To determine the positions of the double bonds and the hydroxyl group,the methyl ester of the hydroxy acid is partially saturated followingthe procedures of Attygalle et al (Tetrahedron Lett., Volume 36, 5471,1195; herein incorporated by reference). After concentration to dryness,the sample is redissolved in approximately 10 μl ethanol, approximately30 μl of about 10% hydrazine in ethanol and approximately 30 μl of about0.6% hydrogen peroxide in ethanol is added, and the solution is heatedto about 60° C. for about 1 hour. After being allowed to cool to aboutroom temperature, about 35 μl of approximately 5% HCl in water is added.The solution is extracted approximately twice with approximately 40 μlGC² hexane and the hexane solution is washed about four times withapproximately 50 μl of water before being analyzed by GC/MS as describedabove in Example 3.

Partial reduction results in a mixture of mono- and di-unsaturatedproducts as established by GC/MS analysis. This mixture is then subjectto ozonolysis following the procedures of Beroza et al., Anal. Chem.,Volume 38, 1976, 1966 and Anal. Chem., Volume 39, 1131, 1967, bothherein incorporated by reference in their entirety. The product isanalyzed by GC/MS as described above in Example 3. GC/MS analysis (CI)shows the presence of three diagnostic GC peaks with (M+1)⁺ ions at m/z187, 229 and 271, corresponding to H(CO)(CH₂)_(n) (CO)OCH₃ with n=7, 10and 13, respectively. Methyl linolenate treated in the same way givesidentical products. Thus, the olefinic bonds in the chain are located oncarbons 9, 12 and 15 and the alcohol group on either the 17th or 18thcarbon.

EXAMPLE 4

To determine the position of the hydroxyl group, the hydroxy acid methylester is dissolved in approximately 50 μl ethyl acetate. About fivemilligrams of Pd₂ O is added and total saturation is achieved by agentle bubbling of H₂ for about 18 hours. GC/MS (EI) analysis (as inExample 3 above) of the product shows an m/z 299 (M-15) and strong m/z270/271 (M-44/M-43) which are conclusive for a hydroxyl group on C17. Apyrrolidide derivative of the saturated methyl ester is preparedfollowing procedures of B.Å. Anderson, Prog. Chem. Fats and otherLipids, Volume 16, 279, 1978; which is herein incorporated by referencein its entirety. GC/MS analysis of the product (as in Example 3 above)shows an m/z 338 (M-15) and m/z 309 (M-44) confirming the C17 locationof the hydroxyl group.

EXAMPLE 5

The following procedure is used to produce the synthetic 17-hydroxylinolenic acid glutamine conjugate.

Racemic 17-hydroxylinolenic acid is synthesized, as illustrated in FIG.5, as follows:

1-Ethoxyethyloxy-8-benzoyloxy-3,6-nonadiyne (1). A mixture containingapproximately 2.3 g (about 12.8 mmol) of the ethoxyethyl ether of3,6-heptadiynol (Huang, W., Pulaski, S. P., and Meinwald, J. J. Org.Chem., Volume 48, 2270 1983; herein incorporated by reference in itsentirety) in about 5 mL of tetrahydrofuran was cooled to about 0° C. andtreated with about 13 mL of an approximately 1M solution ofethylmagnesiumbromide in tetrahydrofuran. After about 1 hour, themixture was treated with approximately 1 mL of acetaldehyde and stirredfor about 1 hour at approximately room temperature. The mixture wastreated with approximately 1 mL of anhydrous pyridine followed by about1.53 mL of benzoyl chloride and stirred for approximately an additionalhour. The mixture was treated with about 10 mL of saturated ammoniumchloride solution and about 15 mL of diethyl ether. The organic phasewas separated, washed with brine, and dried over anhydrous MgSO₄. Afterfiltration and removal of the solvent in vacuo, GC/MS analysis showedone major long retention time peak MS (rel. intensity) 327 (1, M-1), 255(5), 115 (8), 105 (90), 91 (20), 77 (35), 73 (100), and 45 (93).

8-Benzoyloxy-Z,Z-3,6-nonadien-1-ol (II). A mixture of crude I in about100 mL of methanol was treated with approximately 0.3 g ofp-toluenesulfonic acid. After about 3 hours TLC analysis indicatedalmost no starting material remained. GC/MS analysis showed one longretention time component (>95%) MS (rel. intensity) 256 (0.5, M+), 255(3), 226 (1), 134 (5), 133 (2), 105 (100), 91 (12), 77 (35), and 51(20). This mixture was treated with about 5 mL of saturated NaHCO₃, andthe solvent was removed in vacuo. The residue was taken up in ether andpurified through a short silica gel (hexane/ ether approximately 9:1)column to provide approximately 2.7 g of unstable 3,6-nonadiyn-1,8-diol,8-benzoate. A mixture of approximately 1.25 g of this material in about60 mL of ethanol containing approximately 0.32 g of quinoline andapproximately 180 mg of lindlar catalyst was hydrogenated at atmosphericpressure until hydrogen uptake ceased. After about 2 hours,approximately 100 mg of additional catalyst was added and the reactionwas continued until hydrogen uptake ceased (Mori, K, and Ebata, T.,Tetrahedron, Volume 42, 3471,1986; herein incorporated by reference inits entirety). GC/MS analysis showed one major long retention timecomponent: MS (rel. intensity) 260 (1, M+), 229 (1), 138 (15), 123 (2),105 (100), 93 (18), 91 (15), 79 (45), and 77 (50). The product wasisolated by filtration through celite and removal of the solvent invacuo. The residue was taken up in ether, washed with dilute HCl, andbrine, and dried over MgSO₄. The infrared spectrum of a sample of thismaterial isolated by preparative gas chromatography showed importantabsorptions at 3433 (br), 3053, 3012, 1712, 1602, 1580, 1450, 1268,1108, 1043, and 709cm⁻¹.

1-Triphenylphosphonium-8-benzoyloxy-Z,Z-3,6-nonadiene, iodide (III). Amixture containing approximately 2.025 g (approximately 7.8 mmol) of IIin about 50 mL of methylene chloride was treated at about 0° C. withabout 1.6 mL of triethyl amine and about 0.75 mL ofmethanesulfonylchloride, the mixture was stirred about two hours, washedwith an approximately saturated Na HCO₃ solution and dried over MgSO₄.GC/MS analysis showed the presence of a single product, GC/MS (relintensity) 243 (2, M-CH₃ SO₃), 233 (2, M-C₆ H₅ CO), 175 (5), 120 (20),105 (100), 91 (15), 79 (18), 77 (35). After the solvent was removed invacuo, the residue was taken up in about 50 mL of acetone and stirredwith approximately 1.35 g of Nal for about 2.5 hours. The solvent wasremoved in vacuo, the residue was taken up in ether and washed withabout 10% Na₂ S₂ O₃, brine, dried over anhydrous MgSO₄ and the solventremoved in vacuo. GC/MS analysis showed one major component (>95%) MS(rel. intensity) 248 (5, M+-122), 175 (8), 121(12), 105(100), 93(25),79(20), and 77(45). The residue was taken up in about 7 mL ofacetonitrile and treated with approximately 2.0 g of triphenylphosphineand heated to reflux overnight. Removal of the solvent provided anunstable, hygroscopic brittle foam, insoluble in anhydrous ether orbenzene.

Methyl, 17-benzoyloxylinolenate (IV). A mixture containing approximately0.65 g of the powdered brittle foam in about 15 mL of tetrahydrofuranwas cooled to approximately -80° C. under an argon atmosphere, andtreated with about 1.0 mL of an approximately 1M commercial solution ofsodium hexamethyldisilazide (3. Bestmann, H. J., Roth, K., Michaelis,K., Vostrowsky, O., Schafer, H. J., and Michaelis, R., Liebigs Ann.Chem., 417, 1987; herein incorporated by reference in its entirety).After about 15 minutes, a solution containing approximately 0.2 g (1mmol) of methyl-9-oxononanoate (Pennington, F. C., Clemer, W. D.,McLamore, W. M., Bogert, V. V., and Solomons, I. A., J. Am. Chem. Soc.,Volume 75, 109, 1953; Burgstahler, A. W., Weigel, L. O., and Shaefer, C.G. Synthesis, 767, 1976; both incorporated by reference in theirentirety) in about 2 mL of tetrahydrofuran was added and the mixture wasstirred overnight and warmed to approximately room temperature. Afterthe addition of approximately 15 mL of saturated ammonium chloridesolution and approximately 15 mL of ether, the organic phase wasseparated, dried over MgSO₄, and the solvent was removed in vacuo toprovide approximately 0.4 g of a mixture. GC/MS analysis revealed thepresence of the starting oxo-ester, triphenylphosphine oxide, and asingle very long retention time peak (20)%: MS (rel intensity) 290 (5,M-122), 147 (3), 133 (10), 119 (12), 105 (100), 94 (20), 93 (15), 87(5), 81 (15), 79 (25), 78 (27), 77 (30), 74 (5), and 55 (20).

17-Hydroxylinolenic acid (V). The above mixture was stirred with aqueousLiOH, approximately 0.5 M, and tetrahydrofuran at about room temperaturefor about 8 hours. After removal of the solvent in vacuo, the solutionwas extracted with ether, acidified with dilute HCl, and extracted asecond time with ether (about 20 mL). The second ether extract was driedover MgSO₄, filtered, and the solvent removed in vacuo to provideapproximately 0.12 g of residue. GC/MS analysis revealed the majorcomponent which had a mass spectrum MS (rel intensity) 276 (5, M-18),221 (2), 193 (1), 147 (5), 133 (10), 119 (12), 107 (13), 105 (15), 95(10), 94 (15), 93 (13), 91 (25), 84 (20), 81 (20), 80 (30), 79 (33), 69(20), 67 (27), 60 (15), 55 (70), 43 (100), and 41 (80).

The 17-hydroxylinolenic acid is then coupled to glutamine as follows:

Approximately 1.1-1.2 equivalents of glutamine (D- or L-) was exchangedinto its lithium salt on an approximate 5 cm×1 cm ID column packed withAmberlite CG-50 (100-200 mesh, Sigma, St. Louis, Mo.), cation exchangeresin following the procedures described by R. Kramell et al,Tetrahedron, Volume 44, 5791, 1988; herein incorporated by reference inits entirety. The product was dispersed in about 1 to 2 ml of DMF(Dimethyl formamide). About one to two hundred microliters of water wasadded followed by about 1.1-1.2 equivalents of freshly distilledtriethylamine. In a second container, approximately 1 equivalent(approximately 10-50 mg) of 17-hydroxylinolenic acid was dissolved inapproximately 2 ml of DMF followed by approximately 1.1-1.2 equivalentsof HOBT (Hydroxybenzotriazole). To this solution, approximately 1.1-1.2equivalents of EDAC (1-Ethyl-3(Dimethylaminopropyl)carbodiimide) wasadded and the solution stirred for about 1 minute before the contents ofthe first container was added to the second and the mixture stirredovernight at about room temperature. Following the coupling reaction,about 4 ml of water was added and the acidity adjusted to about pH 3with approximately 1 M HCl. The mixture was extracted approximately 3times with distilled water. The organic phase was concentrated to neardryness and the residue was redissolved in about 2 ml of methylenechloride. The methylene chloride fraction was in aliquots of about 0.2ml put on an activated 3 ml 10SPE diol cartridge (Bakerbond, J. T.Baker, Phillipsburg, N.J.). The column was rinsed with a first fractionof about 3 ml methylene chloride followed by approximately 2 ml ofmethanol. The methanol fraction was finally purified on HPLC using anLDC 4100 pump with SM5000 diode array UV detector (LDC Analytical,Riviera Beach, Fla.), monitoring UV detection from about 190 to 360 nm.The reverse phase column (YMC 18 ODS-AQ S-5 200 Å, 46 mm ID×250 mm YMCCO., ltd., Kyoto, Japan) was eluted with a solvent consisting of 25%acetonitrile in water with 0.4 mM ammonium acetate (Aldrich), using aflow of about 1.2 ml/min. The synthetic product had a retention time ofapproximately 26 minutes, which was identical to that of the naturalproduct. The synthetic conjugates also show identical UV absorption.FAB-MS/MS analysis gives a daughter ion spectrum identical to that ofthe natural product. GC/MS analysis of the methylated synthetic hydroxyacid show this to be identical to the methyl ester of the hydroxy acidobtained by methanolysis of the natural product.

EXAMPLE 6

The synthetic compounds are bioassayed with excised corn seedlings asdescribed above in Example 1. Approximately 300 pmol (approximately 135ng) and approximately 900 pmol (approximately 405 ng) of pure syntheticD-Gln and L-Gln forms of the active compound, purified natural product,sodium salts of synthetic 17-hydroxylinolenic acid, D-glutamine,L-glutamine were tested. The results are shown in FIG. 6. The syntheticL-glutamine form is as active as the natural product. The D-glutamineform shows activity at the higher dose. Neither the free hydroxy acid orD- or L-glutamine alone show any biological activity. Approximately 300pmol (approximately 135 ng) and approximately 900 pmol (approximately405 ng) of pure synthetic D-Gln and L-Gln forms of linolenic acid andpurified natural product were tested. The results are shown in FIG. 7.The L-glutamine form of linolenic acid showed more biological activitythan the D-glutamine form, and the biological activity at either dosewas approximately half of that of the natural product. Bioassaying atapproximately 5, 17, 50, 170 and 500 pmol/plant of natural elicitor andthe synthetic N-(17-hydroxylinolenoyl-L-glutamine, shows almostidentical dose response curves. The results are shown in FIG. 8.Therefore, it can be concluded that the most biologically activecompound identified so far is N (17-hydroxy 9, 12, 15octadecatrienoyl)-L-glutamine.

The foregoing detailed description is for the purpose of illustration.Such detail is solely for that purpose and those skilled in the art canmake variations therein without departing from the spirit and scope ofthe invention.

We claim:
 1. A composition for inducing plants to produce and/or releaseinsect attractant volatiles, said composition comprising:an effectiveamount of a compound selected from the group consisting ofN-(17-hydroxylinolenoyl)-L-glutamine, N-(linolenoyl)-L-glutamine, andmixtures thereof, to induce plants to produce and/or release insectattractant volatiles; and a surfactant.
 2. The composition of claim 1,wherein said compound is N-(17-hydroxylinolenoyl)-L-glutamine.